Genome-wide analysis of common carp (Cyprinus carpio) mucin genes and their roles in mucosal immune response following the Aeromonas hydrophila infection

Highlights

• •A set of 11 mucin family genes were identified across the common carp genome.
• •The common carp mucin genes presented tissue-specific expression patterns.
• •The expression of mucin genes in mucosal tissues were regulated after bacterial infection.

Abstract

Common carp is a globally farmed and economically important freshwater fish species. Due to the intensive culture conditions, the farmed common carp are susceptible to various pathogens, causing high mortality and serious economic losses to the carp culture industry. The route of infection is usually through the main mucosal tissues. In order to generate an effective strategy to better manage the fish disease, it is important to understand mucosal immune related genes as well as their expressions during infection. In this study, the common carp mucin gene family were identified and characterized through bioinformatics analysis, and their expression patterns were examined in healthy tissues of common carp as well as the mucosal tissues after the infection of Aeromonas hydrophila. Our results showed that, there were a set of 11 mucin genes across the entire common carp genome. Functional domain prediction and phylogenetic analysis supported their annotation and orthologies. Examining gene copy number across several vertebrates showed that muc4 and muc6 genes were present in other vertebrate, but were lost in teleost fish, which appeared as a result of gene loss after the split of teleost fish from tetrapods during evolution. The mucin genes presented tissue-specific expression patterns in various healthy common carp tissues, with relatively high expression levels in mucosal tissues, indicating their important roles likely in mucosal immune response. Furthermore, the expression of all mucin genes except for muc2-1 were significantly regulated at one or more timepoints in minimum one mucosal tissue after bacterial infection, suggesting that the mucin gene family played critical roles in the mucosal immunity of common carp in response to pathogenic invasion. Collectively, our findings provided fundamental genomic resources for better understanding the vital roles of mucin genes in the defense against pathogen invasion in teleost.

Introduction

The mucosal immune system of vertebrates consists of a unique array of innate and acquired immune cells and molecules that work collaboratively to protect the host from pathogen invasion [1]. In teleost fish, the mucosal immune system is mainly composed of mucosal-associated lymphoid tissues, including intestinal associated lymphoid tissue (GALT), gill associated lymphoid tissue (GIALT), and skin associated lymphoid tissue (SALT) [2]. Fish inhabit in water environment, and constantly directly face a large number of various microorganisms residing in their aquatic environment. The mucosal-associated lymphoid tissues constitute the first defense barrier of fish immunity, which are crucial to protect themselves against pathogens [3]. The mucus that lines the epithelium of mucosal-associated lymphoid tissues plays an important but poorly understood role in protection [4].

Mucin, a kind of macromolecular glycoprotein, is the primary structural component of mucus. So far, more than twenty different mucin genes have been identified in humans and higher vertebrates [5]. Based on their structure and localization, the mucin proteins are classified into two types, the membrane-bound mucins, such as MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC20, MUC21, and the secreted mucins, such as MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC19 [6]. The membrane-bound mucins are retained in plasma membrane due to the presence of a transmembrane domain, and play roles in various signaling pathway. The secreted mucins, usually characterized by the presence of several functional domains such as von Willebrand D (VWD), cysteine rich (C8), and trypsin inhibitor like cysteine rich (TIL) domain, form a protective layer over organs that are in contact with external environment and create a physical barrier against pathogens. In both classes, a protein backbone is studded with glycans that are linked to either the N-Glycans or to the O-glycans. These glycans can influence the behavior and physiology of pathogenic microbes and reduce their ability to spread [7,8].

Mucin genes have been extensively studied in humans and mammals. For instance, the abnormal expression of muc1 gene in tumor tissues makes it a potential biomarker of tumor, and has been used in the diagnosis and biological therapy of tumor [9]. The expression of muc2 gene is decreased in patients with ulcerative colitis (UC) and decreases with the severity of the disease [10]. There is a high expression level of muc5ac gene in respiratory tract of patients with ventilator-associated pneumonia, and excessive secretion of MUC5AC is an important factor in the progression of ventilator associated pneumonia (VAP) [11]. The expression of muc15 in esophageal squamous cell carcinoma tissues was significantly higher than that in normal esophageal tissues, and its abnormally high expression may be related to the occurrence and development of esophageal squamous cell carcinoma [12]. MUC17 inhibits the metastasis of gastric cancer cells [13]. Compared to mammals, knowledge gaps regarding mucins are apparent in fish. Only single or two mucin genes were characterized in a few fish species, and was likely involved in defending pathogen invasion, such as mucin 2 [14] and mucin 5b [[14], [15], [16]].

Common carp, Cyprinus carpio, is a globally farmed and economically important freshwater fish species. Due to the intensive culture conditions, the farmed common carp are more susceptible to pathogens. Aeromonas hydrophila is one of the most common pathogens affecting common carp, which cause high mortality and serious economic losses to the carp culture industry [17]. The route of infection is usually through the main mucosal tissues, such as gill, skin and intestine. In order to generate an effective strategy to better manage the fish disease, it is important to understand mucosal immune related genes as well as their expression during infection. In the present study, the common carp mucin gene family were identified across the entire genome. The gene sequence structure and functional domain were analyzed. In addition, the gene expression patterns were examined in healthy tissues of common carp as well as the mucosal tissues after the infection of A. hydrophila. The systematic study of common carp mucin gene family in this study provided fundamental genomic resources for better understanding the vital roles of mucin genes in the defense against pathogen invasion in teleost.

Materials and methods

Gene identification and sequence analysis

The sequences of each gene member of zebrafish and human mucin family was obtained from the Ensembl and NCBI databases, and used as query sequences to search all available common carp genome resources using an independent BLAST tool with an E-value cutoff of le-10. In order to verify the accuracy of candidate genes, the candidate common carp gene sequences were query sequences by reciprocal BLAST, and the coding sequences were confirmed by BLAST searches against NCBI non-redundant protein sequence database. The web-based simple modular architecture research tool (SMART) was used to predict conserved domain, with default settings.

Phylogenetic analysis

Phylogenetic analysis was performed using protein sequences of mucin gene members from common carp and several other vertebrates, including human (Homo sapiens), mouse (Mus musculus), chimpanzee (Pan troglodytes), zebrafish (Danio rerio), Mozambique tilapia (Oreochromis mossambicus), Japanese medaka (Oryzias latipes), tropical clawed frog (Xenopus laevis), Chinese softshell turtle (Pelodiscus sinensis), great tit (Parus major), chicken (Gallus gallus), coelacanth (Latimeria chalumnae), zig zag eel (Mastacembelus circumcinctus), siamese fighting fish (Betta splendens), channel catfish (Ictalurus punctatus), Indian medaka (Oryzias melastigma), Nile tilapia (Oreochromis niloticus), black rockcod (Hyporthodus ergastularius), Pacific white-sided dolphin (Lagenorhynchus acutus), blue whale (Balaenoptera musculus), retrieved from the NCBI and Ensembl databases. The accession number of each sequence used in this study is shown in supplementary Table S1. Software MEGA 11 was used to construct the phylogenetic tree by using the neighbor-joining method. Bootstrap test with 1000 replicates was conducted to evaluate the phylogenetic tree.

Challenge experiment and sample collection

All sampling procedures involved in treatment and disposal in this study were approved by the Animal Health and Utilization Committee of the Aquatic Genomics Application Center of the Chinese Academy of Fishery Sciences prior to commencement. The fish used in this study were 5-month-old common carp (11 ± 1 cm in length, 16 ± 2 g in weight), from the Fish Breeding Center of the Chinese Academy of Fishery Sciences, Beijing, China. The fish were temporarily reared in the circulating water system for 2 weeks at a temperature of 28 °C and a pH of 7.5–7.75. Healthy common carp were fed regularly at 9 a.m. and 6 p.m., and the water quality test was carried out daily.

The fish were randomly divided into 3 control groups and 3 treatment groups, with 100 fish in each group. By intraperitoneal injection, the treatment group was injected with 0.1 mL of A. hydrophila in sterile PBS (3 × l0⁸ CFU/mL), and the control group was injected with the same volume of sterile PBS. Fish are euthanized with MS-222 before sampling and every effort is made to eliminate suffering. Skin, gill and intestinal tissues were collected at 6 h, 12 h, 24 h and 48 h after the challenge. Six fish were randomly selected from each group at each time point. Four healthy common carp were randomly selected, and 9 tissues including brain, heart, spleen, liver, kidney, intestine, gill, muscle and skin were collected from each fish. All samples collected were immediately immersed in RNAlater™ (Ambion, USA) and stored at −80 °C until the RNA isolation.

Real-time fluorescence quantitative PCR analysis

Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of mucin family genes in 9 healthy tissues including brain, heart, spleen, liver, kidney, intestine, gill, muscle, skin. The expression level of mucin family genes in 3 mucosal tissues including skin, gill, and intestine following the bacterial infection were examined by using qRT-PCR as well. First, the total RNA from each sample was extracted using the RNA Easy Fast Tissue/Cell Kit (Tiangen, China). Second, ReverTra Ace® qPCR RT Master Mix with gDNA Remover was used to synthesize the first strand of cDNA. Finally, qRT-PCR was performed using SYBR Green Master Mix reagent (Transgen Biotech, Beijing, China) on ABI PRISM 7500 real-time detection system (Life Technologies). GAPDH gene was used as reference gene. The initial polymerase was activated at 95 °C for 60 s. 40 cycles were performed at 95 °C for 15 s, 60 °C for 15 s, 72 °C for 45 s. The 15 μL reaction mixture consists of 0.3 μL forward primer, 0.3 μL reverse primer, 7.5 μL SYBR™Green PCR Master Mix, 1 μL cDNA sample (300 ng/μL), and 5.9 μL ddH₂O. A negative control with no template was conducted on all plates. Each sample was repeated in three times to confirm the expression pattern.

The expression level of treated group transcript was determined as a relative expression ratio to control group. All samples were normalized to the expression level of GAPDH in the same sample. For analyzing the expression pattern of genes in healthy tissues, the tissue with the lowest expression value was used as the control group. The p value less than 0.05 was considered significant.

Results and discussion

Identification and sequence analysis of common carp mucin genes

Mucin, as the main component of fish mucus, plays an important role in resisting pathogen infection. Despite their importance, systematic analysis of mucin gene family has been rarely conducted in fish species. As reported, the mucin genes usually contain long segments of highly repetitive sequences, which varies among the mucins and are poorly conserved among species [18]. The large size and the repetitive nature of mucin genes make their identification difficult [19]. In the present study, based on a high-quality whole genome sequences of common carp as well as available transcriptome data, a total of 11 mucin genes were identified across the whole genome, including 6 secreted mucins (muc2-1, muc2-2, muc5ac-1, muc5ac-2, muc5b, muc19) and 5 membrane-bound mucins (muc1-1, muc1-2, muc15-1, muc15-2, muc17). The complete coding sequences of each gene were obtained, and all sequences could be found in the NCBI database with accession number PRJNA510861. Detailed information is shown in Table S2, including gene ID, coding sequence, exon number, and genome location. The number of amino acids encoded by mucin genes ranged from 100 to 2739. The number of exons varied from 3 to 83 among different mucin gene members, but the number of exons between the duplicates of muc1, muc2, muc5, and muc15 were quite similar, respectively (Table S2).

Analysis of physicochemical properties and functional domain of common carp mucin proteins

As shown in Table S3, the prediction of physicochemical properties of common carp mucin protein shows that the molecular weight ranges from 30,530.19 to 645,252.85 KD, with an average molecular weight of 210,225.91 KD. The isoelectric point ranges from 3.92 to 7.57, and the average value is 5.41. The isoelectric points of MUC1-1, MUC1-2 and MUC2-1 were greater than 7.0, indicating these proteins were basic proteins. While the isoelectric points of the remaining proteins were less than 7.0, indicating their acidic properties. Except for MUC1-2 and MUC17, the instability coefficient of mucin proteins were higher than 40, suggesting them being unstable proteins. The aliphatic index ranged from 42.84 to 93.57, and the average index was 63.52. The grand average of hydrophilic (GRAVY) is between −0.866 and −0.034, indicating that they were hydrophilic proteins.

Functional domains were predicted based on protein sequences. As shown in Fig. S1, the domain architecture between membrane-bound mucin and secreted mucin were obviously different. All secreted mucins contain VWD, C8, and TIL, with the number of domain varies among different mucin members. These domains contributed to oligomerization through disulfide bond formation [20], which provides the gel-forming properties of mucus [21]. The domains in membrane-bound mucin were less conserved among different mucin member. The sea urchin sperm protein, enterokinase, and agrin domain proteins (SEA domain), mucin15 domain, and herpes simplex virus type 1 BLLF1 domain (Herpes_BLLF1 domain) was predicted in MUC1, MUC15, and MUC17, respectively (Fig. S1).

Phylogenetic analysis of common carp mucins

To confirm the annotation of common carp mucin genes and further understand their evolutionary relationships, phylogenetic analysis was performed. A phylogenetic tree was constructed by using 46 mucin protein sequences from other 19 representative vertebrates, including 5 mammalian species, 2 bird species, 1 reptile species, 1 amphibian species, and 10 fish species. Overall, our phylogenetic analysis results showed that each of the common carp mucin gene clustered with its respective counterpart from other species, providing additional evidence for the annotation (Fig. 1). All 57 protein sequences formed six major clades, include MUC1 group, MUC2 group, MUC5 group, MUC15 group, MUC17 group, and MUC19 group. The common carp mucins were grouped with their orthologous genes of other fish species, and then clustered with other vertebrates, with strong bootstrap support.

Fig. 1.

Phylogenetic analysis of common carp mucin gene family.

Copy numbers of mucin genes in common carp

The copy number of mucin genes in teleost fish and tetrapods could provide insight into the mucin gene evolution. Our results showed that common carp had multiple copies of each mucin gene including muc1, muc2, muc5 and muc15. Gene member muc3, muc4, muc6, muc7, muc8, muc12, muc13, muc16, muc20, muc21, muc22 were not detected in common carp genome. Based on genomic resources from NCBI and Ensemble genome databases combined literature searches, we next examined the copy numbers of mucin genes in different vertebrates (Table S4). Overall, the copy number of mucin genes in higher vertebrate was more than that in teleost fish. In human, the number of mucin genes was the most, but most of them had only a single copy. It seems that muc4 and muc6 genes were present in other vertebrate, but were lost in teleost fish, which appeared as a result of gene loss after the split of teleost fish from tetrapods during evolution. Comparing to other teleost fish, the total number of mucin genes in common carp was more than most of others. Most of the mucin genes in common carp had multiple homology copies, while only a single copy was found in other fish. The multiple copies of mucin genes in common carp were likely derived from gene duplications in a species specific manner.

Tissue expression of mucin genes in common carp

In order to determine the expression pattern of mucin genes in common carp, qRT-PCR method was performed using gene-specific primers in different healthy tissues. Overall, common carp mucin genes were widely expressed in different tissues but displayed unique tissue-specific expression patterns. For instance, muc1-1, muc2-2, muc5ac-1, muc5b, muc17 genes were relatively highly expressed in skin, liver, heart, gill, intestine, but low expressed in brain, kidney and muscle. Gene muc1-2 was relatively highly expressed in brain, skin, liver, heart, gill, intestine, but low expressed in kidney and muscle. Gene muc2-1 was relatively highly expressed in liver, heart, gill, intestine, but low expressed in brain, skin, kidney and muscle. Gene muc5ac-2 was relatively highly expressed in only gill. Genes muc15-1 and muc19 were expressed at a relatively low level in all tested tissues. Gene muc15-2 was expressed at a relatively high level in all tested tissues. Among all tested tissues, the spleen was the tissue with the lowest expression value, which therefore was used as control group. Interestingly, most mucin genes were relatively highly expressed in the mucosal tissues, including skin, gill and intestine, indicating their critical roles likely in maintaining the function of mucus and in host mucosal immune response.

Expression of mucin genes in common carp mucosal tissues following bacterial infection

To further understand its potential role in mucosal immune response, we examined the expression of mucin gene family in three mucosal tissues of common carp, following the infection of A. hydrophila. Our results showed that the expression of all common carp mucin genes except for muc2-1 were significantly different in minimum one mucosal tissue following the bacterial infection, but differed in the timing, tissues and extent/direction of regulation (Fig. 2). Gene muc1-1 was significantly up-regulated in intestine and skin after 6 h post infection. Gene muc1-2 was significantly down-regulated in intestine at 12 h post infection. Gene muc2-2 was significantly down-regulated in intestine after 12 h post infection, and up-regulated in gill at all tested time-points. Gene muc5ac-1 was significantly up-regulated in skin at 12 h post infection. Gene muc5ac-2 was significantly up-regulated in intestine after 12 h post infection. The expression of muc5b and muc15-1 were significantly regulated in intestine, skin and gill after infection. Gene muc15-2 were significantly up-regulated in gill at 6 h, but down-regulated after 12 h post infection. Gene muc17 was significantly down-regulated in intestine after 12 h post infection, but significantly up-regulated in gill after 6 h post infection. Gene muc19 was significantly down-regulated in all three mucosal tissues, but at different timepoints. These results indicated that the mucin gene family played important roles in the mucosal immunity of common carp in response to pathogenic invasion. Further studies are needed to conclusively prove the molecular mechanism of downstream regulation.

Fig. 2.

The temporal expression analysis of mucin genes after A. hydrophlia infection in common carp mucosal tissues. Asterisks indicate statistical significance at the level of p < 0.05.

In summary, the mucin family genes of carp were systematically analyzed for the first time. A set of 11 mucin genes were identified and annotated across the common carp genome. Phylogenetic analysis provided evidence for the homology of these genes. Combined with the domain prediction and phylogenetic tree, muc2, muc15 and muc19 are highly conserved among bony fish. The mucin gene family are widely expressed in common carp, especially with high expression levels in mucosal tissues. To further explore the roles of mucin genes in host mucosal immune response against A. hydrophila infection, the temporal and spatial expression of mucin genes were detected. Taken together, our results indicated that common carp mucin genes were involved in common carp mucosal immunity in defense of bacterial infection. However, further detailed studies are needed for verifying the functions of each gene involving in the mucosal immune response. Our findings could contribute to a better understanding of the immune response of mucin genes in teleost fish, and may suggest effective strategies to better manage the fish disease and provide useful information for future physiological, immunological and comparative genomics studies in common carp and other fish species.

CRediT authorship contribution statement

Jiali Wang: Writing – original draft, Software, Methodology, Data curation. Qi Zhou: Validation, Software, Methodology. Yanliang Jiang: Writing – review & editing, Supervision, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.